Microtubules

Microtubules play a role in

1. Organization of intracellular organelles and transport

of vesicles (motor proteins)

2. movement of cilia and flagella

3. Nerve cell, red blood cell and flagellar structure

4. Alignment and separation of chromosomes during

mitosis and meiosis

microtubule structure

a hollow tube made of a subunit dimer of 1 alpha and 1 beta tubulin

-they connect slightly out of phase, causing the low pitch spiral (hollowness)

microtubules are made up of subunits called

tubulin dimers which is a dimer of 1 alpha tubulin and 1 beta tubulin

which of the 3 types is in a constant state of flux between growth and shrinkage called dynamic instability?

microtubules

at the plus end of the microtubule

the beta subunit is exposed

at the minus end of the microtubule

the alpha subunit is exposed

which end of the microtubule grows faster?

the positive end

what does the natural affinity between tubulin dimers cause?

rapid exponential growth into a polymer with a predictable pattern if concentration of tubulin dimers is high

when tubulin is first added to a test tube

the polymerization first has a lag period and then spontaneous polymerization

when tubulin polymerization reaches equillibrium

the growth stabilizes but the dynamic instability still exists

-because of this, reactions can move in either direction

what is the rate limiting/slowest step of the tubulin experiment?

the lag phase, even if you add super high concentration

during the lag phase, microtubules exist as

individual dimers

(nucleation)

during the elongation phase, microtubules are

growing as dimers connect together

(exponential/log growth)

at the plateau phase, the microtubule is

experiencing growth and shrinkage

(equillibrium)

the concept of critical concentration

if the concentration of tubulin is too low, microtubule formation will get trapped in the lag phase and wont proceed

what is unique about the different ends of a microtubule?

they are unique in structural properties and rates of polymerization

the negative end growth

is possible but requires a much higher initiation concentration and still once it starts will grow slower than the positive end

rate of shrinkage is

not dependent on concentration

the leading factor in tubulin dynamics

GTP

-the beta tubulin dimers contain GTP and can sometimes form a cap which will bend the structure and cause disassembly

which part of the microtubule dimer hydrolyses GTP to GDP?

only the beta subunit

GTP bound dimers are considered to be

activated causing a higher affinity for the microtubule, a lower critical concentration, and a faster rate of reaction

the hydrolysis from GTP to GDP will cause

a reduced affinity

catastrophe

the microtubule releases the GDP tubulin (Shrinking)

catastrophe is caused by

loss of the GTP cap, exposing the GDP lattice

rescue

the microtubule grows by addition of GTP tubulin

Microtubule organizing centers

any structure used to nucleate and organize microtubules

-centrosomes for example but also basal bodies for cillia

the primary MTOC in animals

the centrosome

singlets

a group of 13 protofilaments

-most common microtubule form in the body

example of a singlet

cytoplasm

doublet

an A ring with 13+ a B ring with 10 protofilaments

doublets example

cillia and flagella

triplets

13+10+10

-basal bodies and centrioles

gamma tubulin complex

-a preformed template for the subunits to attach and grow from

the portion of the MTOCs that nucleate microtubules

-is attached to the side of another microtubule

how do gamma tubules work?

by lowering the critical concentration, making it easier for the tubule to grow

which end of the microtubule is anchored into the gamma ring?

the negative end

which end of the microtubule will shrinkage and elongation most likely occur at?

the positive end

what are the two centralized MTOCs all eukaryotic cells use during mitosis?

spindle and poles

centrosome during interphase

-2 centrioles, 90 degrees apart

-both contains 2 rings (13+10)

-also uses a basal body with 3 rings (13+10+10)

cell during mitosis

microtubules come from the spindle poles, everything is replicated

-plus ends are the farthest away from the MTOC

in nerve cells, centralized microtubules are only found in

developing neuron

-matured nerve cells only have decentralized

what is unique about the positive end of nerve microtubules?

they can be pointing toward the cell body unlike in other cells

as nerve cells mature, centrosomes

no longer are used as the primary MTOC, instead, decentralized ones in the axons and dendrites are used

nerve cell microtubules are not dynamic

true

experimental proof of microtubule polarity

cells were cooled and given a drug to depolymerize the microtubules

-when the cell is rewarmed, the microtubule begins to reform and grow from the plus end

-the conclusion that the negative ends are in the MTOC, and the tubule grows from the positive side

colchicine and synthetic colcemid

bind between alpha and beta subunits and causes depolymerization

-at small doses, it can restabilize

toxol

binds to the sides of the tubule and stabilizes it

-can inhibit chromosome segregation

scientists use high concentrations of colchicine to

depolymerize all microtubules and test the cells ability to perform phagocytosis without them

how are colchicine and taxols used as anticancer agents?

they disrupt or stabilize microtubules which determines cell division

-no cell division, no cancer replication

the 4 categories of proteins that control and regulate microtubules

1. Maps

2. Tips

3. proteins for microtubule disassembly

4.motor proteins

plus end tracking proteins (+TIPS)

EB1 and EB3

Creation of a cap to prevent depolymerization and encourage growth at the + end

-also connects tubules to other structures

destabilizing proteins

Kinesin 13

tubulin dimer sequestration and GTP hyrdolysis is done by

stathmin

motor proteins

use the energy from ATP hydrolysis to move along the filament

ie. Kinesin and dyneins

MAPs (microtubule associated proteins)

consist of two domains, one negative to interact with microtubule surfaces and one positive to interact with amino acids

-separated by a right angle as a spacer between them

the spacing between microtubules in ____ containing cells is greater than in ____ containing cells

MAP2, tau

-both contain the same number of microtubules but MAP2 specifically is used to widen the caliber

positively charged amino acids from MAP2 interact with ____

the negatively charged microtubule surface

the negative domain of the MAP2

sticks out at a right angle for a spacer

using immunofluorescence, microtubules show up as ____ and +TIPs show us as ___

red, green

microtubule growth most oftenly is at the ____ end. However, during the cell cycle, depolymerization must occur here instead

plus

proteins used for microtubule break down

kinesin 13 and stathmin

kinesin 13

it interacts with tubulin subunits at the plus side, causing it to bend and disassemble

stathmin

prefers to act on the minus end, causing the tubule to shrink at the plus end

microtubule associated motor proteins

use kinesins and dyneins to move in different directions on the microtubule

kinesins can move

vesicles and portions of ER toward the plus end (cell periphery)

dyneins can move

endosomes and lysosomes towards the minus end (the cell center) for nucleus invasion

structure of cytosolic kinesins

globular heads for ATP hydrolysis and interaction with microtubules

two heavy chains in a alpha helix manner

several light chains for vesicle binding

kinesin 1

transportation

kinesin 2

- heterotrimeric: 2 heavy chains that are different

also transportation

kinesin 5

dual head

-organization and elongation

motor activity for kinesins

1. P release from the lagging head causes weak binding to the MT

2. ATP binding to the leading head causes a tension force

3. The tension from step 2 pushes the lagging head to the front, making it the new leading head

4. Tight binding and releasing of an ADP for ATP hydrolysis, a P is released and steps are repeated

structure of dynein (not axoneme specific)

a heavy chain consisting of a stem, a linker, a stalk that acts as a foot, and 6 AAA ATPase repeats creating a coiled coil stalk

axonemal dyenins

non cystolic, contain flagella or cilia for movement

flagella

only 1 per cell, single cell eukaryotes

use of a whipping motion

cilia

many per cell, assist in moving particles and fluids across the cell surface

a structural component of a cilia or flagella

axoneme

axoneme

an arrangement of doublets made of A and B tubules, called the 9+2 arrangement because 9 doublets are connected with a pair of singlet microtubules in the center

nexin proteins

connect doublets to eachother by linking A tubule to B tubule

structure for axoneme dyenins

a protein composed of outer and inner dynein’s.

• A base is permanently attached to the A tubules 13 MT

• a head area that detaches and rebinds to the B tubules 10 MT

  • only movement on the B tubule, causing the axoneme bending force

the head of the axoneme dynein is interacting with the

the B tubule

the base of the dyenin always interacts with the

A tubule

what allows for the extreme stabillity of the axoneme?

the nexin linkers between the doublets that completely prevent movement, as the dyneins try to move, it bends causing the wavelike motion for flagellar function

if you remove the nexin linker

bending will stop and the two flagella begin to slide

nexin linkers are removed by

cleaving via protease

intraflagellar transport (IFT)

if you want to move towards the positive end, you can’t do that with dyenin. Instead, they attach to cargo carried by Kinesin-2 to get there. If they want to go back to the end, they hitchhike with dyenin

when particles cannot move to the + end, how do they get there?

Kinesin-2

when particles cant move towards the - end, how do they get there?

cytoplasmic dyenin

movement of molecules back to the tip help _____ whereas movement of molecules to the base helps ____

create new flagella, remove flagella

Primary cillium

-the cells sensory organelle

-allows the cell to respond to stimuli

polycystic kidney disease

-affects 1/1000

-formation of cysts in the kidneys leading to failure

-defects in urine flow sensingn

normally cases of polycystic kidney disease are caused by mutation of which genes?

PC1 protein (w PKD1 gene) and PC2 protein (PKD2 gene)

Badet Beidl Syndrome

-cognitive impairment, retinal degeneration, polydactyly, and hypogonadism

-sensory receptors are not being transported to the cilia by intraflagellar transport

what are some root causes of Bardet Biedl syndrome?

1. A mutation has caused an issue with the BBsome

   • Because of this, normal amounts of kinesin and dynein, but the BBSome prevents cargo from attaching and therefore, material is not getting moved to the primary cilia